Name: microfluidic buffer exchange for interference-free micro/nanoparticle cell engineering
Uploaded: 2016-07-10T15:00:00
Description: Watch this Scientific Journal Video about Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering at JoVE.com

Kind gift of THP-1 cells from Dr. Mark Chong and assistance in microfabrication from Dr. Yuejun Kang and Dr. Nishanth V. Menon (School of Chemical and Biomedical Engineering, Nanyang Technological University) were greatly acknowledged. This project was funded by NTU-Northwestern Institute of Nanomedicine (Nanyang Technological University). H.W.H. was supported by Lee Kong Chian School of Medicine (LKCMedicine) postdoctoral fellowship.

1. Nanoparticles (NPs) Labeling of Mesenchymal Stem Cells and Monocytes
<ol>
	<li>Culture mesenchymal stem cells (MSCs) in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics to &#8805;80% confluency prior to labeling. Similarly, culture THP-1 cells (ATCC) in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS to a density of ~106 cells/ml.</li>
	<li>Load silica NPs (~500 &#181;m) with calcein dye solution (200 &#181;M) usi...

<ol>
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G., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+inertial+focusing,+ordering,+and+separation+of+particles+in+microchannels.">Continuous inertial focusing, ordering, and separation of particles in microchannels.</a> Proc. Natl. Acad. of Sci USA. 104, 18892-18897 (2007).</li><li>Hur, S. C., Henderson-MacLennan, N. K., McCabe, E. R. B., Di Carlo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformability-based+cell+classification+and+enrichment+using+inertial+microfluidics.">Deformability-based cell classification and enrichment using inertial microfluidics.</a> Lab Chip. 11, 912-920 (2011).</li><li>Mach, A. J., Di Carlo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+scalable+blood+filtration+device+using+inertial+microfluidics.">Continuous scalable blood filtration device using inertial microfluidics.</a> Biotechnol. Bioengin. 107, 302-311 (2010).</li><li>Gossett, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inertial+manipulation+and+transfer+of+microparticles+across+laminar+fluid+streams.">Inertial manipulation and transfer of microparticles across laminar fluid streams.</a> Small. 8, 2757-2764 (2012).</li><li>Amini, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+fluid+flow+using+sequenced+microstructures.">Engineering fluid flow using sequenced microstructures.</a> Nat. Commun. 4, 1826 (2013).</li><li>Sollier, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inertial+microfluidic+programming+of+microparticle-laden+flows+for+solution+transfer+around+cells+and+particles.">Inertial microfluidic programming of microparticle-laden flows for solution transfer around cells and particles.</a> Microfluid. Nanofluid. 19, 53-65 (2015).</li><li>Lee, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivariate+biophysical+markers+predictive+of+mesenchymal+stromal+cell+multipotency.">Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency.</a> Proc. Natl. Acad. of Sci USA. 111, E4409-E4418 (2014).</li><li>Hou, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+retrieval+of+circulating+tumor+cells+using+centrifugal+forces.">Isolation and retrieval of circulating tumor cells using centrifugal forces.</a> Sci. Rep. 3, 1259 (2013).</li><li>Hou, H. W., Bhattacharyya, R. P., Hung, D. T., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+and+drug-resistance+profiling+of+bacteremias+using+inertial+microfluidics.">Direct detection and drug-resistance profiling of bacteremias using inertial microfluidics.</a> Lab Chip. 15, 2297-2307 (2015).</li><li>Yeo, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference-free+Micro/nanoparticle+Cell+Engineering+by+Use+of+High-Throughput+Microfluidic+Separation.">Interference-free Micro/nanoparticle Cell Engineering by Use of High-Throughput Microfluidic Separation.</a> ACS Appl. Mater. Inter. 7, 20855-20864 (2015).</li><li>Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+Lithography.">Soft Lithography.</a> Angew. Chem. Int. Edit. 37, 550-575 (1998).</li><li>Lee, W. 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The DFF cell purification technology described herein enables rapid and continuous separation of labeled cells in a high throughput manner. This separation approach is ideal for large sample volume or high cell concentration sample processing, and is better than conventional membrane-based filtration which is prone to clogging after extended use. Similarly, affinity-based magnetic separation requires additional cell labelling steps which are laborious and expensive. The purified cells are shown to retain their labeled ag...

Engineering cells by agent-loaded micro/nanoparticles (NPs) is a simple, genomic integration-free, and versatile method to enhance bioimaging capability and augment/supplement its native therapeutic properties in regenerative medicine.1-3 Cellular modifications are achieved by labeling the plasma membrane or cytoplasm with an excess concentration of agent-loaded NPs to saturate the binding sites. However, a major drawback of this method is the significant quantities of unbound particles remaining in solution after cell labeling processes, which can potentially confound precise identification of particle-engineered cells or complicate therapeutic outcomes.4,5 In addition, exposure to NPs containing transformative agents (growth factors, corticosteroids, etc.) at excessively high concentration can cause cytotoxicity and misdirected exposure may induce unintended consequences on non-target cells. Even particulate carriers comprising of &#34;biocompatible&#34; materials [e.g., poly(lactic co-glycolic acid), PLGA] can incite potent immune cell responses under certain conditions as well.6 This is especially risky in individuals with impaired immunity (e.g., rheumatoid arthritis) which potentially delays systemic nanoparticle clearance.7 Thus, efficient removal of free particles prior to the introduction of particle-engineered cells is of great importance to minimize toxicity profile and reduce misdirected exposure to agent-loaded particles in vivo.
Conventional gradient centrifugation is often used to separate engineered cells from free particles but is laborious and operated in batch mode. Moreover, shear stresses experienced by cells during high-speed centrifugation and the constituents of the density gradient medium may compromise cell integrity and/or influence cell behavior.8 Microfluidics is an attractive alternative with several separation technologies including deterministic lateral displacement (DLD)9, dieletrophoresis10,11 and acoustophoresis12 developed for small particles separation and buffer exchange applications. However, these methods suffer from low throughput (1-10 &#181;l&#183;min&#8722;1) and are prone to clogging issues. Active separations such as dielectrophoresis-based methods also require differences in intrinsic dielectrophoretic cell phenotypes or additional cell labeling steps to achieve separation. A more promising approach involves inertial microfluidics &#8212; the lateral migration of particles or cells across streamlines to focus at distinct positions due to dominant lift forces (FL) at high Reynolds number (Re).13 Due to its high flow conditions and superior size resolution, it has often been exploited for size-based cell separation14,15 and buffer exchange applications.16-18 However, buffer exchange performance remains poor with &#8764;10&#8722;30% contaminant solution as the separated cells usually remain close to the boundary between original and new buffer solutions.16-18 More importantly, the size distribution of target cells has to be similar to achieve precise inertial focusing and separation from the original buffer solution which poses an issue especially in the processing of heterogeneous-sized cell types such as mesenchymal stem cells (MSCs).19
We have previously developed a new inertial microfluidics cell sorting technique termed Dean Flow Fractionation (DFF) for isolating circulating tumor cells (CTCs)20 and bacteria21 from whole blood using a 2-inlet, 2-outlet spiral microchannel device. In this video protocol, we will describe the process of labeling THP-1 (human acute monocytic leukemia cell line) suspension monocytic cells (~15 &#181;m) and MSCs (10-30 &#181;m) with calcein-loaded NPs, followed by fabrication and operation of the DFF spiral microdevice for efficient recovery of labeled cells and removal of unbound NPs.22 This single step purification strategy enables continuous recovery of labeled suspension and adherent cells suspended in fresh buffer solution without centrifugation. Moreover, it can process up to 10 million cells&#183;ml&#8722;1, a cell density amenable for regenerative medicine applications.

<table border="0" cellpadding="0" cellspacing="0" width="781">
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	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:780px;">Cell lines &#38; Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Mesenchymal Stem Cells (MSCs)</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">PT-2501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">12-614F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">THP-1 monocyte cells (THP-1)</td>
			<td style="width:127px;">ATCC</td>
			<td style="width:119px;">TIB-202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Roswell Park Memorial Institute (RPMI) 1640 media</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">12-702F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Name</td>
			<td style="width:127px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:780px;">Reagents &#38; Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">0.01% poly-L-lysine (PLL)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">3 mL Syringe</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">302113</td>
			<td>Syringe 3ml Luer-Lock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">60 ml Syringe</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">309653</td>
			<td>Syringe 60ml Luer-Lock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:127px;">Biowest</td>
			<td style="width:119px;">P6154-100GR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Calcein, AM (CAM)</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">C1430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Calcein</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Isopropanol</td>
			<td style="width:127px;">Fisher Chemical</td>
			<td style="width:119px;">#P/7507/17</td>
			<td>HPLC Grade 2.5 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Phosphate-Buffered Saline (PBS)</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">17-516Q/12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Plain Microscope Slides</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">FIS#12-550D</td>
			<td>75 X 25 X 1 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:127px;">Dow Corning</td>
			<td style="width:119px;">SYLGARD&#174; 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Scotch tape</td>
			<td style="width:127px;">3M</td>
			<td style="width:119px;">21200702044</td>
			<td>18mm x 25m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Silica NPs (&#8764;200 &#956;m)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">748161</td>
			<td>Pore size 4 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Syringe Tip</td>
			<td style="width:127px;">JEC Technology</td>
			<td style="width:119px;">7018302</td>
			<td style="width:167px;">23GA .013 X .25</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Tygon Tubing</td>
			<td style="width:127px;">Spectra-Teknik</td>
			<td style="width:119px;">06419-01</td>
			<td>.02X.06&#34; 100</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Name</td>
			<td style="width:127px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:780px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Biopsy punch</td>
			<td style="width:127px;">Harris Uni-Core</td>
			<td>69036-15</td>
			<td>1.50 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dessicator</td>
			<td style="width:127px;">Scienceware</td>
			<td style="width:119px;">111/4 IN OD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">High-speed Camera</td>
			<td style="width:127px;">Phantom&#160;</td>
			<td style="width:119px;">V9.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Inverted phase-contrast microscope&#160;</td>
			<td style="width:127px;">Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Plasma cleaner</td>
			<td style="width:127px;">Harrick Plasma</td>
			<td style="width:119px;">PDC-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Syringe Pump</td>
			<td style="width:127px;">Chemyx</td>
			<td style="width:119px;">CX Fusion 200</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic buffer exchange for interference-free micro/nanoparticle cell engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

After labeling the cells with bio imaging agent-loaded NPs overnight, the labeled cells (containing free particles) are harvested and purified by DFF spiral microdevice to remove free NPs in a single step process (Figure 1A). The 2-inlet, 2-outlet spiral microchannel is designed by engineering software and microfabricated using SU-8 photoresist. The patterned silicon wafer is then used as a template for PDMS replica molding using soft lithography techniques (Figur...

Watch this Scientific Journal Video about Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering at JoVE.com

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

The overall goal of this procedure, is to demonstrate a convenient, efficient, and high throughput microfluidic purification technology, to separate free nanoparticles from cells, following cell engineering with the nanoparticles. The major want of this technology is to allow the quick and efficient removal of unbound nanoparticles After the cell laboring procedure, we gather up all our ses-fa-tion. This microfluidic cell purification technology achieves efficient size based separation due to the differential inertial focusing effects of cells and nanoparticles in a spiral micro-device.It can process up to 10 million cells per mil concentration which is very useful for regenerative medicine, as well as latch volume cell processing. This technology is high desired for cell engineering field, which often using nanoparticle to labor cells, for imaging purpose or function control. Demonstrating the procedure will be Hui Min Tay, a research assistant from my laboratory, as well as Doctor David Yeo, a post doctor research fellow from Professor Xi's laboratory.Culture mesenchymal stem cells to greater than 80%confluency on a six well plate prior to labeling the cells with the nanoparticles. Similarly culture THP-1 cells to a density of one million cells per milliliter. Next, dissolve one milligram of silica nanoparticles with one milliliter of two hundred micromolar calcium dye solution.And stir the particles overnight. Also, fabricate PLGA calcium AM nanoparticles using methods described in the reference shown here. Mix either one milligram of the PLGA calcium AM nanoparticles or 150 micrograms of the calcium silica nanoparticles in 01%poly-l-lysine solution in water by pipetting the solution up and down for one minute.And then let it incubate at room temperature for 15 to 20 minutes. Next centrifuge the nanoparticles. Remove the excess poly-l-lysine supernatant and re-suspend the nanoparticles in one milliliter of the appropriate culture medium for the cell type to be labelled.Add 1 milligrams of the labelled nanoparticles to one milliliter of medium for every one million cells in culture. Pipette the mixture of cells, nanoparticles, and medium thoroughly for one minute. And then incubate the mixture for approximately 24 hours.Once labelled, dissociate and harvest the stem cells, and re-suspend them to a concentration of between 100, 000 and 1, 000, 000 cells per milliliter for microfluidic processing. Use labelled THP-1 cells at the same concentration. In order to fabricate the microfluidic spiral device, first prepare a silicone wafer master mold using standard microfabrication techniques.Then, mix 30 grams of the base PDMS prepolymer and three grams of a curing agent thoroughly in a weighing boat. Degas the mixture under vacuum for 60 minutes to remove any air bubbles. Pour the PDMS mixture onto the master mold, patterned with the spiral channel design, carefully to a height of five to 10 millimeters.Then degas the mixture for 60 minutes to remove any air bubbles. Repeat this process until all bubbles are eliminated. Next, place the wafer in an oven, and ensure that the wafer is not tilted during curing, to ensure a constant device height.Cure the PDMS at 80 degrees Celsius for two hours. Once cool, cut out the PDMS spiral device using a scalpel and carefully peel the PDMS slab from the master mold. Then, use a scalpel to trim the edges of the device to ensure a smooth surface for bonding.Next, use a 1.5 millimeter biopsy punch to create two holes for the inlets, and two holes for the outlets. Wash the device with isopropanol to remove any debris. And dry the device in an 80 degree Celsius oven for five minutes.Then, use masking tape to clean the bottom surface of the PDMS device, and one side of a glass slide. Carefully place the glass slide and PDMS device into a plasma chamber with the clean surfaces exposed. Switch on the plasma power to maximum, lower the chamber pressure until the chamber turns pink in color, and expose the components for 60 seconds.Then, remove the slide and PDMS device from the chamber. And bond them together by pressing the plasma exposed surfaces tightly together. Ensure no bubbles are trapped between the two surfaces.Heat the bonded device on a hot plate set at 80 degrees celsius for two hours to strengthen the bond. Cut two pieces of tubing 15 to 20 centimeters in length for the inlets, and attach a 23 gauge needle on one end of each tube. Then, cut two pieces of the same tubing that are five to 10 centimeters in length for the outlets, and attach them to the outlet holes of the device.Prior to running the sample, manually prime the device with a syringe containing 70%ethanol until it flows from the outlet tubing. Allow the ethanol to sit in the device for at least 30 seconds to sterilize it. Then, load 30 milliliters of filtered PBS with 1%BSA into a 60 milliliter syringe.Also, load three milliliters of labelled cells into a three milliliter syringe and check that no air bubbles are trapped in either syringe. Remove any air bubbles by gently ejecting a few drops of liquid out of the nozzle. Connect the inlet syringe tips and tubing to the syringes and secure the syringes to the syringe pumps.Insert the tubes into their respective inlets of the device, and ensure that there are no bubbles along the tubing. With the fluidics now set up, mount the device onto an inverted phase contrast microscope for real time imaging during the cell sorting process. Secure a small waste beaker and two 15 milliliter tubes close to the device on the microscope stage.Place the outlet tubing from both of the device's outlets into the waste beaker. Now, set the flow ratio for sample to sheath buffer to one to 10 by setting the flow rate of the sample syringe to 120 microliters per minute and the sheath syringe to 1, 200 microliters per minute. Run the device for one and a half minutes to give the flow rate a chance to stabilize.Use a high speed camera to confirm the presence of inertially focused cells near the channel inner wall under bright field with phase contrast. Once steady state flow is achieved, and the device is running correctly, position the outlet tubes into different vials to collect separate eluents from the cell outlet and waste outlet. This device is able to correctly sort a labelled suspension of THP-1 monocytic cells from fluorescent silica nanoparticles.High separation efficiencies were obtained as confirmed by both high speed imaging and flow cytometry analysis. The single step purification strategy enables continuous recovery of a labelled suspension and adherent cells that are suspended in fresh buffer solution without the need for centrifugation. The device is capable of high efficiency separation with both silica and PLGA nanoparticles and can separate up to 10 million cells per milliliter with the same high efficiency.After watching this video, you should have a good understanding of how to label cells with nanoparticles. And how to purify the labelled cells by removing excess unbound particles using our specialized microfluidic device.

microfluidic-buffer-exchange-for-interference-free-micronanoparticle

Research

JoVE Journal

Bioengineering

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

Site-specific Bacterial Chromosome Engineering: &amp;#934;C31 Integrase Mediated Cassette Exchange (IMCE)

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&amp;#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.